A major challenge to biomedical science in the coming years will be to understand the principles underlying assembly and function of the macromolecular complexes. Such complexes include those involved in signal transduction, DNA replication, transcription, and protein synthesis, to name a few, and are central to the function of living cells. The mechanistic analysis of molecular assembly by measuring the individual binding or enzymatic events are limited by available methods. Flow cytometry offers sensitive multiparameter fluorescence detection combined with the sub-second kinetic resolution required to analyze complex macromolecular assemblies. Within the past year we have started to develop methods to extend flow technology to the in vitro study of macromolecular assembly and function. The first result of this research is the detailed kinetic analysis of a DNA repair enzyme. Using a microsphere-bound substrate, we designed a real-time kinetic flow cytometric assay to measure DNA cleavage by human flap endonuclease-1 (FEN-1). This novel approach enabled the experimental separation of the binding and cleavage events, and a mechanistic analysis not possible by existing methods. In addition to providing a detailed description of enzyme function, our results provided a mechanistic basis for the design and interpretation of site-directed mutagenesis. This system represents one example of the potential impact of flow cytometry on the study of molecular structure and function. The goals of this project are to extend this microsphere-based flow cytometric approach to the study of other types of molecular interactions. This will be accomplished by developing new instrumentation, calibration approaches, and methods of sample preparation to support a wide range of molecular assembly applications. Specifically, we will 1) develop a flow cytometer capable of on-line DNA melting to support the study of DNA-protein interactions and demonstrate the utility in DNA melting, nicking (by Topo I), and annealing applications; 2) develop strategies and procedures for attaching molecules to microspheres and calibrating their concentrations; 4) examine protein/protein assemblies based on the attachment and calibration strategies; and 5) characterize phospholipid bilayers as novel substrates for assembly on microspheres. We will use these new tools to support several collaborations in the mechanistic analysis of molecular interactions. The instrument development, calibration protocols, and sample preparation techniques described here should have a major impact on the study of macromolecular assemblies.